Calcium-activated chloride currents (ICl.Ca) have been recorded in cardiac muscle cells and play a role in the repolarization of the cardiac action potential. To date, there is no information as to the gene encoding the channel underlying this conductance. Bestrophins are a novel family of proteins that have recently been described to function as calcium-activated chloride channels. The overall goal of our research is to understand the mechanisms by which Bestrophin ion channels mediate cardiac cell physiology. As a first step, we have identified the Bestrophin channel isoforms expressed in mouse heart (RT-PCR, Northern blot, immunofluorescence and immunoblot). We have isolated and cloned the Bestrophin transcripts from mouse heart and performed a preliminary characterization of the current generated from one Bestrophin isoform (mBest-3). The following specific aims are proposed to achieve the overall goal: Specific Aim 1 examines whether Bestrophin proteins function as calcium-activated chloride channels. We will determine the electrophysiological properties of Bestrophin channels (in vitro expression, patch clamp) and investigate which specific isoforms participate in the calcium activated chloride current in mouse cardiac cells. Specific Aim 2 examines the tissue and cellular localization of Bestrophin proteins in heart. We will examine membrane localization of Bestrophin isoforms (confocal microscopy/immunofluorescence) and possible association with accessory proteins (immunoprecipitation, pull-down assays, mass spectrometry), including interactions with other Bestrophins. Specific Aim 3 will evaluate the roles of Bestrophin channels in regulating cardiac cell physiology in vivo, particularly in the context of whole organisms. We will delineate the relative contribution of specific Bestrophin channels in cardiac excitability in mutant mice (targeted gene disruption). To complete these aims, the PI will employ established as well as new models and methods including: cell biology, cellular imaging, pull down assays, electrophysiology, gene knock-out in transgenic mice and cardiovascular phenotyping techniques. The information gained from the proposed study will greatly enhance our understanding the role of calcium-activated chloride channels in cardiac excitability and will provide potential areas for therapeutic targets. PUBLIC HEALTH RELEVANCE: The main trust of this project is to elucidate the function of Bestrophin channel proteins in regulating cardiac excitability especially during repolarization of the cardiac action potential. The proposed studies may provide novel insights into our understanding of the role of Bestrophin channels in cardiac physiology and disease states and thus provide potential areas for therapeutic targets.